Optical microsystem for spectroscopy signals extraction applied to gastrointestinal dysplasia detection

Tese de Doutoramento em Engenharia BiomédicaThe early detection of gastrointestinal cancer, in the dysplastic stage, is essential to increase the patient survival rate. Spectroscopic techniques, particularly diffuse reflectance and fluorescence, can improve the gastrointestinal dysplasia detection, since these techniques can be used to extract biochemical and morphologic information related with the status of a gastrointestinal tissue. Several research groups have developed prototypes for the extraction of diffuse reflectance and fluorescence signals applied to gastrointestinal cancer detection. Despite their advantages associated with the gastrointestinal cancer identification, they have several disadvantages related with the use of complex, high-cost and sophisticate components such as xenon lamps, lasers, monochromators, optical fibers and high quantum efficiency detectors, which may hamper their wide use as well as their huge clinical value. Therefore, it is of utmost importance to develop a low-cost, miniaturized and minimal invasive microsystem for spectroscopic signals extraction. As a result, in this work it is proposed the implementation of a microsystem, which comprises in a single chip, an optical filter system for selection and extraction of the diffuse reflectance and fluorescence signals in relevant spectral bands, a silicon photodiodes matrix (4×4) and its readout electronics, and miniaturized light emitting diodes. The main applications of this microsystem are: its use as a portable device in a surgery room for inspection of total removing of the cancerous or dysplastic tissue; and its integration with the standard endoscopes and colonoscopes using it as an auxiliary, to the physician, in early gastrointestinal cancer detection. Along this thesis, important steps towards that microsystem implementation were achieved. In a first step experimental measurements were performed, with phantoms representative of the main absorbing, scattering and fluorescence properties of gastrointestinal tissues (containing hemoglobin, polystyrene microspheres to represent collagen fibers, and the fluorophores NADH and Carbostyril 124, the latter representing collagen), in order to study the diffuse reflectance and fluorescence typical spectra and their temperature dependence. Moreover, the viability of using only 16 spectral bands (between 350 and 750 nm) for signals extraction was discussed, proving the feasibility of an optical filter system implementation in the final microsystem. Therefore, it were designed, fabricated and characterized 16 MgO/TiO2 and SiO2/TiO2 based thin-film optical filters. Their characterization performed through optical transmittance, selectivity, profilometry and scanning electron microscopy, allowed understanding the deviations between the simulated characteristics and the ones experimentally obtained. Moreover, the optical filters results showed transmittances ranging from 50% to 90% approximately, and a full width half maximum (FWHM) averaging from 11 nm to 20 nm, which fits the required application. The fabricated optical filters had some deviations considering their simulated characteristics, which can be explained by the complexity of the optical filters design, for example, the materials refractive index dependence with wavelength and thin-film thickness. The diffuse reflectance and fluorescence signals that pass through the optical filters can be measured with an on-chip silicon photodetectors matrix (4×4), based on n+/p-epilayer junction photodiodes with an active area of 100 × 100 µm2, and a light-to-frequency converter, per each photodiode, that enables producing a digital signal with a frequency proportional to the photodiode current. As a result, the design and implementation of a CMOS microsystem comprising these components were performed. The photodiodes characterization showed a responsivity of 200 mA/W at 550 nm, approximately, and the light-to-frequency converter connected to the photodiode showed a linear response (R2>0.99) with a sensitivity of 25 Hz/nA at 550 nm, approximately. The behavior of the current-to-frequency converter, with an external current source directly injected in its input, was also studied allowing to confirm its linearity in the range of currents produced in this application, its power consumption of 1 mW, and its maximum input current, approximately 300 µA. This CMOS approach avoids the need of an expensive readout optical microsystem, since it is possible to integrate the photodiodes and the readout electronics in a small silicon area (275 × 100 µm2 per photodiode and its respective converter). The performance of the implemented microsystem and the fabricated optical filters was evaluated, using phantoms (also containing hemoglobin, polystyrene microspheres, NADH and Carbostyril 124). The obtained results have shown the viability of the microsystem (including the optical filter system) to extract diffuse reflectance and fluorescence signals. Some issues were noted on the sensitivity of the implemented optical setups for the on-chip measurements. However, some solutions were proposed for the remaining problems, specifically the future use of miniaturized light emitting diodes and the direct deposition of the optical filters on the top of the photodetection system. Finally, the direct integration of optical filters on top of the photodiodes was discussed and a new approach was tested.The author, Sara Filomena Ribeiro Pimenta, was supported by the Portuguese Foundation for Science and Technology (in portuguese FCT – Fundação para a Ciência e a Tecnologia) with the PhD grant SFRH/BD/87605/2012. This work is also funded by FEDER funds through the “Eixo I do Programa Operacional Fatores de Competitividade” (POFC) QREN, project reference COMPETE: FCOMP-01-0124-FEDER- 020241 and by FCT, project reference PTDC/EBB-EBI/120334/2010. Finally, the author thanks to the PEst-C/FIS/UI0607/2013, UID/FIS/04650/2013, UID/EEA/04436/2013 and POCI-01- 0145-FEDER-006941 for the use of equipment.A deteção precoce do cancro gastrointestinal, na fase de displasia, é essencial para o aumento da taxa de sobrevivência do paciente. As técnicas de espetroscopia, particularmente a refletância difusa e a fluorescência, permitem melhorar a deteção de displasia gastrointestinal, ao poderem ser utilizadas para a extração de informação bioquímica e morfológica associada ao estado do tecido gastrointestinal. Diversos grupos de investigação têm desenvolvido protótipos para a extração de sinais de refletância difusa e de fluorescência, para aplicação na deteção do cancro gastrointestinal. Apesar das vantagens associadas com a identificação do cancro gastrointestinal, esses sistemas apresentam várias desvantagens relacionadas com a utilização de componentes complexos, de elevado custo e sofisticados, como por exemplo, lâmpadas de xénon, lasers, monocromadores, fibras óticas e detetores de elevada eficiência, que podem dificultar a sua ampla utilização, bem como o seu elevado valor clínico. Portanto, é de extrema importância o desenvolvimento de um microssistema de baixo custo, miniaturizado e minimamente invasivo para a extração de sinais de espetroscopia. Assim, neste trabalho é proposta a implementação de um microssistema, num único chip, compreendendo: um sistema de filtros óticos para a seleção dos sinais de refletância difusa e de fluorescência em bandas espetrais relevantes; uma matriz de fotodíodos de silício (4×4) e a respetiva eletrónica de leitura; e díodos emissores de luz miniaturizados. As principais aplicações deste microssistema são: a sua utilização como sistema portátil numa sala de cirurgia para inspeção da remoção total do tecido maligno ou displásico; ou a sua integração com os sistemas de endoscopia e colonoscopia, servindo como auxiliar de diagnóstico, na deteção precoce de cancro gastrointestinal. Com a realização desta tese foram dados passos importantes para a implementação desse microssistema. Numa primeira fase, foram realizados testes experimentais, com um grupo de fantomas representativos das propriedades de absorção, difusão e de fluorescência dos tecidos gastrointestinais (contendo hemoglobina, microesferas de polistireno representando as fibras de colagénio, e os fluoróforos NADH e Carbostyril 124, este último para representar o colagénio), de forma a obter os espetros típicos de refletância difusa e de fluorescência e a influência da temperatura do fantoma nos mesmos. Para além disso, a viabilidade de usar apenas 16 bandas espetrais (entre 350 e 750 nm) para a extração dos sinais espetroscópicos foi discutida, provando a exequibilidade da implementação de um sistema de filtros óticos no microssistema final. Assim, foram desenhados, fabricados e caracterizados 16 filtros óticos baseados em filmes finos de MgO/TiO2 e SiO2/TiO2. A sua caracterização do ponto de vista da transmitância ótica, seletividade, profilometria e microscopia eletrónica de varrimento, permitiu perceber os desvios verificados entre as características simuladas e as obtidas experimentalmente. Para além disso, os resultados da caracterização dos filtros óticos mostraram transmitâncias óticas que variam entre 50% e 90%, aproximadamente, e uma largura a meia-altura (FWHM) média entre 11 nm e 20 nm, o que é adequado para a aplicação pretendida. Os filtros óticos fabricados possuem alguns desvios das suas características simuladas, o que pode ser explicado pela complexidade no projeto de filtros óticos, por exemplo, a dependência dos índices de refração com o comprimento de onda e espessura do filme fino. Os sinais de refletância difusa e fluorescência que atravessam os filtros óticos podem ser medidos através de uma matriz de fotodetetores de silício (4×4), baseada em fotodíodos do tipo n+/p-epilayer com uma área ativa de 100 × 100 µm2, e um conversor luz-frequência, um por cada fotodíodo, que permite produzir um sinal digital com uma frequência proporcional à corrente gerada pelo fotodíodo. Assim, o projeto e a implementação de um microssistema CMOS incluindo esses componentes foram executados. A caracterização dos fotodíodos da matriz resultou num valor de responsividade de 200 mA/W a 550 nm, aproximadamente, e a do conversor luz-frequência, quando ligado a um fotodíodo, resultou numa resposta linear (R2>0.99) com uma sensibilidade de 25 Hz/nA a 550 nm, aproximadamente. O comportamento do conversor corrente-frequência, com uma fonte de corrente externa diretamente injetada na sua entrada, foi também estudado, permitindo confirmar a sua linearidade na gama de correntes envolvidas nesta aplicação, a sua potência de consumo de 1 mW, e a sua corrente de entrada máxima, aproximadamente 300 µA. Esta abordagem em tecnologia CMOS evita a utilização de um microssistema ótico de leitura de elevado custo, uma vez que torna possível a integração dos fotodíodos e respetiva eletrónica de leitura numa área de silício pequena (275 × 100 µm2 por fotodíodo e respetivo conversor). Foi avaliado o desempenho do microssistema implementado e dos filtros óticos fabricados usando fantomas (mais uma vez contendo hemoglobina, microesferas de polistireno, NADH e Carbostyril 124). Os resultados obtidos provaram a viabilidade do microssistema (incluindo o sistema de filtros óticos) para a extração de sinais de refletância difusa e de fluorescência. Foram notados alguns problemas na sensibilidade dos setups óticos implementados para as medições on-chip. No entanto, foram também propostas algumas soluções para os respetivos problemas, especificamente o uso futuro de díodos emissores de luz miniaturizados e a deposição direta dos filtros óticos no sistema de fotodeteção. Finalmente, a integração dos filtros óticos depositados diretamente em cima dos fotodíodos foi discutida e uma nova abordagem foi testada

LTCC packaging for Lab-on-a-chip application

LTCC -pakkaus Lab-on-a-chip -sovellukseen. Tiivistelmä. Tässä työssä suunniteltiin, valmistettiin ja testattiin uusi pakkaustekniikka ”Lab-on-a-chip” (LOC) -sovellukseen. Pakkaus tehtiin pii-mikrosirulle, jolla voidaan mitata solujen kiinnittymistä sirun pintaan solujen elinkelpoisuuden indikaattorina. Luotettavuustestaukset tehtiin daisy-chain -resistanssimittauksilla solunkasvatusolosuhteissa. Lisäksi työssä selvitettiin LTCC- ja ”Lab-on-a-chip” -teknologioiden perusteet teoreettiselta pohjalta. Mikrosirun pakkauksessa käytettiin joustavaa LTCC-teknologiaa. Sähköisiin kontakteihin ja niiden suojauksiin käytettiin sekä johtavia että eristäviä epoksi-liimoja. LOC-sovelluksiin on tärkeää kehittää uusia pakkausmenetelmiä jotta näiden laitteiden kaikki ominaisuudet saadaan toimimaan luotettavasti. Pakkaus testattiin samoissa olosuhteissa missä sitä tullaan käyttämään ja pakkaus kesti kaikki nämä haasteet. Lisäksi esitetty valmistusprosessi on sellainen, että sitä voidaan käyttää myös muihin ”Lab-on-a-chip” -sovelluksiin.Abstract. This work presents design, manufacturing and testing of new packaging method for Lab-on-a-chip (LOC) application. Packaging was made for silicon microchip which can measure cell adhesion on chips surface as indication of cell viability. Reliability testing was done with daisy-chain resistance measurement in real conditions. Moreover basic theory of LTCC and Lab-on-a-chip technology is presented. Resilient LTCC technology was used for packaging material and conductive/insulating epoxies were applied for electrical contacts and barriers against the environment. It is fundamentally important to develop new packaging methods for LOC applications, so all the properties can be utilized reliably. Packaging was tested under the cell growth conditions and the package showed to withstand all these challenges. Moreover the presented packaging method is possible to use also in other Lab-on-a-chip applications

Towards Large Scale CMOS Single-Photon Detector Arrays for Lab-on-Chip Applications

Single-photon detection is useful in many domains requiring time-resolved imaging, high sensitivity and high dynamic range. In this paper the miniaturization and performance potential of solid-state single-photon detectors are discussed in the context of lab-on-chip applications where high accuracy and/or high levels of parallelism are suited. Technological and design trade-offs are discussed in view of recent advances in integrated LED matrix technology and the emergence of new multiplication based architectures

A Label Free CMOS-Based Smart Petri Dish for Cellular Analysis

RÉSUMÉ Le dépistage de culture cellulaire à haut débit est le principal défi pour une variété d’applications des sciences de la vie, y compris la découverte de nouveaux médicaments et le suivi de la cytotoxicité. L’analyse classique de culture cellulaire est généralement réalisée à l’aide de techniques microscopiques non-intégrées avec le système de culture cellulaire. Celles-ci sont laborieuses spécialement dans le cas des données recueillies en temps réel ou à des fins de surveillance continue. Récemment, les micro-réseaux cellulaires in-vitro ont prouvé de nombreux avantages dans le domaine de surveillance des cellules en réduisant les coûts, le temps et la nécessité d’études sur des modèles animaux. Les microtechniques, y compris la microélectronique et la microfluidique,ont été récemment utilisé dans la biotechnologie pour la miniaturisation des systèmes biologiques et analytiques. Malgré les nombreux efforts consacrés au développement de dispositifs microfluidiques basés sur les techniques de microscopie optique, le développement de capteurs intégrés couplés à des micropuits pour le suivi des paramètres cellulaires tel que la viabilité, le taux de croissance et cytotoxicité a été limité. Parmi les différentes méthodes de détection disponibles, les techniques capacitives offrent une plateforme de faible complexité. Celles-ci ont été considérablement utilisées afin d’étudier l’interaction cellule-surface. Ce type d’interaction est le plus considéré dans la majorité des études biologiques. L’objectif de cette thèse est de trouver des nouvelles approches pour le suivi de la croissance cellulaire et la surveillance de la cytotoxicité à l’aide d’un réseau de capteurs capacitifs entièrement intégré. Une plateforme hybride combinant un circuit microélectronique et une structure microfluidique est proposée pour des applications de détection de cellules et de découverte de nouveaux médicaments. Les techniques biologiques et chimiques nécessaires au fonctionnement de cette plateforme sont aussi proposées. La technologie submicroniques Standard complementary metal-oxide-Semiconductor (CMOS) (TSMC 0.35 μm) est utilisée pour la conception du circuit microélectronique de cette plateforme. En outre, les électrodes sont fabriquées selon le processus CMOS standard sans la nécessité d’étapes de post-traitement supplémentaires. Ceci rend la plateforme proposée unique par rapport aux plateformes de dépistage de culture cellulaire à haut débit existantes. Plusieurs défis ont été identifiés durant le développement de cette plateforme comme la sensibilité, la bio-compatibilité et la stabilité et les solutions correspondantes sont fournies.----------ABSTRACT High throughput cell culture screening is a key challenge for a variety of life science applications, including drug discovery and cytotoxicity monitoring. Conventional cell culture analysis is widely performed using microscopic techniques that are not integrated into the target cell culture system. Additionally, these techniques are too laborious in particular to be used for real-time and continuous monitoring purposes. Recently, it has been proved that invitro cell microarrays offer great advantages for cell monitoring applications by reducing cost, time, and the need for animal model studies. Microtechnologies, including microelectronics and microfluidics, have been recently used in biotechnology for miniaturization of biological and analytical systems. Despite many efforts in developing microfluidic devices using optical microscopy techniques, less attention have been paid on developing fully integrated sensors for monitoring cell parameters such as viability, growth rate, and cytotoxicity. Among various available sensing methods, capacitive techniques offer low complexity platforms. This technique has significantly attracted attentions for the study of cell-surface interaction which is widely considered in biological studies. This thesis focuses on new approaches for cell growth and cytotoxicity monitoring using a fully integrated capacitive sensor array. A hybrid platform combining microelectronic circuitry and microfluidic structure is proposed along with other required biological and chemical techniques for single cell detection and drug discovery applications. Standard submicron complementary metal–oxide–semiconductor (CMOS) technology (TSMC 0.35 μm) is used to develop the microelectronic part of this platform. Also, the sensing electrodes are fabricated in standard CMOS process without the need for any additional post processing step, which makes the proposed platform unique compared to other state of the art high throughput cell assays. Several challenges in implementing this platform such as sensitivity, bio-compatibility, and stability are discussed and corresponding solutions are provided. Specifically, a new surface functionalization method based on polyelectrolyte multilayers deposition is proposed to enhance cell-electrode adherence and to increase sensing electrodes’ life time. In addition, a novel technique for microwell fabrication and its integration with the CMOS chip is proposed to allow parallel screening of cells. With the potential to perform inexpensive, fast, and real-time cell analyses, the proposed platform opens up the possibility to transform from passive traditional cell assays to a smart on-line monitoring system

Lab-on-CMOS Sensors and Real-time Imaging for Biological Cell Monitoring

Monitoring biological cell growth and viability is essential for in vivo biomedical diagnosis and therapy, and in vitro studies of pharmaceutical efficacy and material toxicity. Conventional monitoring techniques involve the use of dyes and markers that can potentially introduce side effects into the cell culture and often function as end-point assays. This eliminates the opportunity to track fast changes and to determine temporal correlation between measurements. Particularly in drug screening applications, high-temporal resolution cell viability data could inform decisions on drug application protocols that could lead to better treatment outcomes. This work presents development of a lab-on-chip (LoC) sensor for real-time monitoring of biological cell viability and proliferation, to provide a comprehensive picture of the changes cells undergo during their lifecycle. The LoC sensor consists of a complementary metal-oxide-semiconductor (CMOS) chip that measures the cell-to-substrate coupling of adherent cells that are cultured directly on top. This technique is non-invasive, does not require biochemical labeling, and allows for automated and unsupervised cell monitoring. The CMOS capacitance sensor was designed to addresses the ubiquitous challenges of sensitivity, noise coupling, and dynamic range that affect existing sensors. The design includes on-chip digitization, serial data output, and programmable control logic in order to facilitate packaging requirements for biological experiments. Only a microcontroller is required for readout, making it suitable for applications outside the traditional laboratory setting. An imaging platform was developed to provide time-lapse images of the sensor surface, which allowed for concurrent visual and capacitance observation of the cells. Results showed the ability of the LoC sensor to detect single cell binding events and changes in cell morphology. The sensor was used in in vitro experiments to monitor chemotherapeutic agent potency on drug-resistant and drug-sensitive cancer cell lines. Concentrations higher than 5 μM elicited cytotoxic effects on both cell lines, while a dose of 1 μM allowed discrimination of the two cell types. The system demonstrates the use of real-time capacitance measurements as a proof-of-concept tool that has potential to hasten the drug development process

Integrated CMOS Capacitance Sensor And Microactuator Control Circuits For On-Chip Cell Monitoring

"Cell Clinics," CMOS/MEMS hybrid microsystems for on-chip investigation of biological cells, are currently being engineered for a broad spectrum of applications including olfactory sensing, pathogen detection, cytotoxicity screening and biocompatibility characterization. In support of this effort, this research makes two primary contributions towards designing the cell-based lab-on-a-chip systems. Firstly it develops CMOS capacitance sensors for characterizing cell-related properties including cell-surface attachment, cell health and growth. Assessing these properties is crucial to all kinds of cell applications. The CMOS sensors measure substrate coupling capacitances of anchorage-dependent cells cultured on-chip in a standard in vitro environment. The biophysical phenomenon underlying the capacitive behavior of cells is the counterionic polarization around the insulating cell bodies when exposed to weak, low frequency electric fields. The measured capacitance depends on a variety of factors related to the cell, its growth environment and the supporting substrate. These include membrane integrity, morphology, adhesion strength and substrate proximity. The demonstrated integrated cell sensing technique is non-invasive, easy-to-use and offers the unique advantage of automated real time cell monitoring without the need for disruptive external forces or biochemical labeling. On top of the silicon-based cell sensing platform, the cell clinics microsystem comprises MEMS structures forming an array of lidded microvials for confining single cells or small cell groups within controllable microenvironments in close proximity to the sensor sites. The opening and closing of the microvial lids are controlled by actuator hinges employing an electroactive polymer material that can electrochemically actuate. In macro-scale setups such electrochemical actuation reactions are controlled by an electronic instrument called potentiostat. In order to enable system miniaturization and enhance portability of cell clinics, this research makes its second contribution by implementing and demonstrating a CMOS potentiostat module for in situ control of the MEMS actuators

Microsystem Based on CMOS Multielectrode Array for Extracellular Neural Stimulation and Recording

Neurobiology is constantly in search of new tools and techniques to extract structural and functional information from neural circuitry. Conventional electrophysiological stimulation and measurement technique such as patch clamping have become the standard techniques for accurate stimulation and recording of electrical activities in neurons. Nevertheless, the number of electrodes that can be introduced into the working chamber is severely limited by the electrode dimension and head stages. Integrating electrodes on chip with complementary metal-oxide-semiconductor (CMOS) technologies enables significantly higher throughput, making analysis on large neural networks possible. This thesis presents the design, characterization, verification, and post-fabrication steps of a microsystem based on a fully integrated high-density multielectrode array (MEA) chip for extracellular stimulation of neural activity. The active MEA is implemented in a standard 0.25 μm CMOS technology with 65,536 non-Faradaic electrodes in an array area of 9 mm2. Each electrode can be configured to produce unique stimulus waveform, delivering a spatial resolution exceeding 12 μm and a temporal resolution exceeding 125 nsec. The array is integrated with neurons in both dispersed culture and acute thalamocortical slices. Experimental results verify the array functionality by attaining high-resolution stimulation of dispersed primary hippocampal neuronal cultures. Neuronal activity induced from stimulation is detected through changes in real-time calcium fluorescence calibrated with cell-attached patching. Precise electrical stimulation of individual neurons is achieved by optimizing stimulation waveforms, culture preparation, and interface design. The design of a second MEA CMOS chip that integrates extracellular recording with on-chip stimulation is also presented. The chip contains 256x256 non-Faradaic circular electrodes with 14 μm diameter and 20 μm pitch. The active area of the array at 32 mm2 is designed to accommodate entire mouse thalamocortical acute slice with an electrode density of 2000 electrodes per square milimeter. Each electrode integrates with a stimulation pulse generator and a single-transistor transconductance amplifier. The new configuration does not require optical recording and reduces the mechanical setup of the microsystem